肠球菌为主要菌群的腹泻标本的16S rRNA与PFGE分析

The fecal bacterial community in diarrheal samples explored by 16S rRNA and PFGE analysis

目的了解肠球菌为主要菌群的腹泻标本的微生态及分离肠球菌菌株的克隆特征。方法根据细菌16S rRNA保守区序列设计通用引物,以腹泻病人粪便标本中的总DNA为模板,PCR扩增16S rRNA片段。将获得的片段克隆入T载体进行测序,与数据库中发表的序列进行比对,判断标本中细菌的种类和比例,并对每份标本分离的各50株肠球菌选择20株进行PFGE分析。结果对4份标本的多次16S rRNA序列分析表明,该4份标本均以Enterococcus faecium为主(所占比例>68%),PFGE结果显示每个病人分离屎肠球菌菌株是克隆性的(一份标本除外)。结论从16S rRNA和PFGE的角度对腹泻粪便标本进行了分析,得到腹泻儿童肠球菌分布的基本特征,其致病性和相关机理还待今后实验范围的进一步扩大和研究的深入。

The purpose of this research was to understand fecal micro-flora of Enterococcus-related diarrhea feces and the clone characteristics of isolated Enterococcus strains.Primer was designed according to 16S rRNA gene and integrated DNA extracted from patients＇ fecal samples was used as the template to amplify the conserved sequence of 16S rRNA by PCR.After the PCR product was purified and cloned into T vector to sequence,PFGE was used to analyze 20 out of 50 Enterococcus isolates from each specimen.The 16S rRNA gene sequencing analysis indicated that these 4 portions of diarrhea feces were dominated by Enterococcus faecium（〉68%） and the isolates were clonal except only one portion.Although the basic flora characteristics of diarrhea feces could be revealed by 16S rRNA gene sequencing analysis and PFGE analysis,the pathogenicity and mechanism of Enterococcus were still waiting for further experimental exploration